2110 Swapna et al.
Asian J. Chem.
6.29. FT-IR (KBr, νmax, cm-1): 3427.37(N-H str.), 3073.51 (C-H
arom. str.), 2926.09 (C-H aliph. str.), 1609.41 (C=O str.), 1519.88
(C=C str.), 1012.65 (C-Cl str.); 1H NMR (CDCl3, δ ppm): 1.90
(s, 3H, CH3), 6.18 (s, 1H, benzylidene), 6.50-7.25 (m, 3H,Ar-H),
8.13 (s, 1H, NH); MS (m/z): 255 [M]+, 257 [M+H]+.
Val-339 and Val-332. Binding modes of the studied pyrazo-
lones were found to be similar to that of celecoxib and most
of them were found to interact with Phe-504 amino acid residue.
Pyrazolones possessing phenylurea or phenylthiourea, interact
with different amino acid residues (π-π stacking of phenyl
ring with Trp-387 and Tyr-385) indicating that pyrazolones
affinity for COX-2 enzyme is influenced/effected with substi-
tution [27].
As the title compounds exhibited good binding affinity
towards the target enzyme, we have designed few more pyrazo-
lones by introducing different functional groups such as 4-Cl,
3,4-Cl2, 4-NO2, 3,4-(OCH3)2 and 3,4,5-(OCH3)3 on benzylidene
ring (M5-M9) and performed molecular docking studies, to
study the importance of substitution on benzylidene ring.
Among methoxy containing derivatives, 3,4-dimethoxy
benzylidene derivative (M8) exhibited good binding affinity
(∆G = -7.96 kcal/mol) indicated that position of methoxy groups
is critical for binding at the active site of receptor. 4-Chloro
and 3,4-dichloro substituted derivative and 4-nitro derivatives
(M5, M6 and M7) showed moderate interactions (∆G = -7.56
and -7.57 kcal/mol) when compared to unsubstituted derivative
(M1) (∆G = -7.37 kcal/mol), suggesting that presence of halogen
increases binding affinity. Binding mode of celecoxib and deriv-
ative M8 with COX-2 is shown in Fig. 1.
P2X3 receptors: The purinergic receptor P2X ligand-
gated ion channel 3 (P2X3) is highly expressed by nociceptive
sensory neurons and involved in pain processing and inflam-
mation. When ATP binds to P2X3 ion channel, it leads to an
increase in Ca2+ concentration ultimately membrane depolari-
zation. The development of ligands that selectively activate or
block specific P2X receptor subtypes represents a promising
strategy to develop new drugs for the treatment of pain, cancer,
inflammation and neurological diseases [28,29].
4-Hydroxy benzylidene derivative (M2) showed highest
binding interaction with -7.18 kcal/mol indicated that hydrogen
binding ability is favourable for the binding interaction which
is comparable to the standard drugATP (-8.55 kcal/mol). Unsubsti-
tuted derivative (M1) showed poor interaction towards this
receptor (-6.77 kcal/mol), which was found to increase with
the presence of either electron releasing substituent groups or
electron withdrawing substituent groups on benzylidene ring
indicating that substituent groups on benzylidene ring favours
binding. Among the compounds, 3,4-dimethoxy (M8), 3,4-
trimethoxy derivative (M9) showed good binding affinity
(-6.97 and -6.99 kcal/mol) indicating the importance of methoxy
groups on benzylidene ring. Halogen bearing derivatives, 4-nitro
derivative and 4-dimethylamino derivatives exhibited moderate
interactions with the target receptor. Pyrazolones were found
to be located at the loop region (Leu249-Cys256) and most of the
compounds were able to establish hydrogen bonding with NH
of Asp-248 with oxygen atom present in the pyrazolone ring.
Binding mode of ATP and derivative M3 with P2X3 receptor
is shown in Fig. 2.
4-(4-Nitrobenzylidene)-3-methyl-1H-pyrazol-5(4H)-
one (M7): m.f. C11H9N3O3, m.p. 182-184 ºC; Elemental analysis
(Found) %: C, 57.19; H, 3.90; N, 18.12; O, 20.70. FT-IR (KBr,
ν
max, cm-1): 3453.37(N-H str.), 2998.02 (C-H arom. str.),
2826.09 (C-H aliph. str.), 1615.58 (C=O str.), 1575.45 (C=C
str.), 1428.73 (N-O assym. str.), 1227.48 (N-O sym. str.); 1H NMR
(CDCl3, δ ppm): 1.92 (s, 3H, CH3), 6.18 (s, 1H, benzylidene),
6.59-7.54 (m, 4H,Ar-H), 8.11 (s, 1H, NH); MS (m/z): 232 [M+H]+.
4-(3,4-Dimethoxybenzylidene)-3-methyl-1H-pyrazol-
5(4H)-one (M8): m.f. C13H14N2O3, m.p. 216-218 ºC; Elemental
analysis (Found) %: C, 63.47; H, 5.71; N, 11.36; O, 19.47.
FT-IR (KBr, νmax, cm-1): 3392.67 (N-H str.), 2960.87(C-H arom.
str.), 2840.13 (C-H aliph. str.), 1656.06 (C=O str.), 1622.66
(C=C str.); 1H NMR (CDCl3, δ ppm): 1.90 (s, 3H, CH3), 3.02 (s,
6H, (OCH3)2, 6.17 (s, 1H, benzylidene), 6.46-7.48 (m, 3H, Ar-H),
8.09 (s, 1H, NH); MS (m/z): 202 [M]+, 205 [M+H]+.
3-Methyl-4-(3,4,5-trimethoxybenzylidene)-1H-pyrazol-
5(4H)-one (M9): m.f. C14H16N2O4, m.p. 209-211 ºC; Elemental
analysis (Found) %: C, 60.87; H, 5.82; N, 10.13; O, 23.17.
FT-IR (KBr, νmax, cm-1): 3405.27(N-H str.), 2909.04 (C-H arom.
str.), 2836.77 (C-H aliph. str.), 1667.70 (C=O str.), 1597.94
(C=C str.); 1H NMR (CDCl3, δ ppm): 1.88 (s, 3H, CH3), 3.08
(s, 9H, (OCH3)3, 6.18 (s, 1H, benzylidene), 6.35-7.62 (m, 2H,
Ar-H), 8.08 (s, 1H, NH); MS (m/z): 206 [M+H]+.
Molecular docking studies: 3-Methyl-4-substituted benzy-
lidene pyrazol-5-ones were previously reported to possess anti-
nociceptive activity. SAR revealed that presence of 4-hydroxy
(M2)/2,3-dimethoxy (M3)/4-dimethylamino groups (M4) on
benzylidene ring increase the potency of the parent structure
(M1) [14]. Molecular docking studies were performed to predict
affinities and binding modes of these derivatives towards few
nociceptive receptors (COX-2, P2X3, TRPV1 and glutamate).
The objective of the study was to identify the effect of substi-
tuent groups on binding affinity.
COX-2 Enzyme: COX-2, inducible form of cyclooxyge-
nase (COX) enzyme is a validated target for the development of
antiinflammatory and antinociceptive agents. COX-2 enzyme
contain large solvent accessible surface in the active site; amino
acids such as Val-523, Arg-513, Arg-120, Tyr-355 and Glu-
524 are situated around the active site.
Results of the molecular docking studies revealed that 2,3-
dimethoxy benzylidene derivative (M3) showed most favou-
rable interaction with ∆G of -7.79 kcal/mol whereas unsubsti-
tuted derivative (M1) showed poor interaction with ∆G of -7.37
kcal/mol indicating that methoxy substitution is favourable for
binding at the COX-2 enzyme. 4-Dimethylamino or 4-hydroxy
benzylidene derivatives (M2 and M4) showed ∆G of -7.59 kcal/
mol. Results were compared using celecoxib which showed
highest binding affinity with ∆G of -10.15 kcal/mol. Celecoxib
was found to establish hydrogen bonding with amino acid residue
Phe-504 andArg-499. The pyrazolones were found to be located
at the loop region (Arg-499-Pro-514) and arylidene ring was
found to be embedded by amino acids such as Ala-327, Phe-42,
TRPV1 ion channel: TRPV1or vanilloid receptor 1 can
be activated by eicosanoids, capsaicin, protons and resinifera-
toxin [27]. It is expressed by primary afferent sensory neurons
of the pain pathway and plays pivotal role in nociception and
neurogenic inflammation [30-32]. Results of the molecular